Innate immune responses are the first line of defense against sexually transmitted diseases (STDs). Initiation of the innate response is based upon recognition of pathogen-associated molecular patterns by host proteins known as toll-like receptors (TLRs). TLR agonists have been identified and have been utilized for modulation of immune responses in humans in the context of vaccine adjuvants. In this project, we propose to test the hypothesis that TLR agonists will prove to be a novel class of topical microbicides providing protection against STDs. Topical microbicides are products designed to protect against STDs. Used vaginally or rectally, microbicides offer women the advantage that they may be "female-controlled". The term microbicide implies that the product kills microorganisms; this is somewhat misleading since microbicides may work in a variety of ways including disrupting the pathogen's membrane or envelope, blocking the receptor-ligand interactions essential for infectivity, inhibiting the intra- or extra-cellular replication of the pathogen, or by altering the vaginal environment or enhancing local immune responses to reduce susceptibility. As proof of concept, we have established the utility of a TLR9 agonist as a microbicide that is effective against HSV-2 genital infections in mice and guinea pig models. This microbicide candidate was effective when applied as a single intravaginal dose within the time frame of 2d prior through 6h after vaginal challenge with HSV-2. To extend our findings and test our hypothesis, in Aim 1 we will determine the typical pattern of TLR gene expression in human vaginal and cervical epithelial cells and select a set of TLR agonists that target the commonly expressed TLRs that then will be assessed for activity in primary cultures of these cells against HSV-2 and C. trachomatis infection. TLR agonists will be prioritized for evaluation based on the in vitro results and then will be evaluated for microbicide potential in mouse models of HSV-2 and C. trachomatis vaginal infections in Aim 2. Those compounds that show greatest microbicide potential will be evaluated mechanistically in Aim 3. Completion of the studies will provide substantial information about the innate immune responses of the vaginal mucosa and will identify TLR agonist microbicide candidates.